Broad spectrum inhibitors

Abstract

The invention features a method of designing broad spectrum inhibitors using structural data, compositions having broad spectrum activity, and methods for treating disease using those compositions.

Description

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the field of inhibitors and methods for identifying or designing broad spectrum therapeutics for use in the treatment of infectious diseases and cancers, particularly where drug resistance is, or could reasonably predicted to be, an obstacle to successful long term therapy.

[0003] The development of drug resistance is one of the most common causes of drug failure in the treatment of diseases involving replicating biological entities (i.e., cancer and infectious diseases). Drug resistance often results from a reduction in drug-binding affinity and can be quantified by the ratio of drug binding affinity (Kd) for variant and wild type target proteins. Administration of a drug introduces a selective pressure upon the replicating biological entity. The result is the emergence of drug resistant strains.

[0004] Drug resistance is a major obstacle to the successful treatment of many cancers and infections, both bacterial and viral. For example, increased resistance of bacterial infections to antibiotic treatment has been extensively documented and has now become a generally recognized medical problem, particularly with nosocomial infections. See, for example, Jones et al., Diagn. Microbiol. Infect. Dis. 31:379 (1998); Murray, Adv. Intern. Med. 42:339 (1997); and Nakae, Microbiologia 13:273 (1997).

[0005] Drug resistance has complicated the treatment for HIV as new mutant strains of HIV have emerged that are resistant to multiple, structurally diverse, experimental and chemotherapeutic antiretrovirals, including HIV protease inhibitors (PIs), nucleoside and non-nucleoside HIV reverse transcriptase inhibitors (NRTIs and NNRTIs), and HIV fusion inhibitors (FIs).

[0006] More than 60 million people have been infected by HIV in the last two decades, and 20 million people have died from HIV/AIDS. While the development of highly active antiretrovirals to treat HIV/AIDS has led to significant reductions in the mortality and morbidity of AIDS, the rapid emergence and spread of drug-resistant mutant strains of HIV is rendering current drugs ineffective, and is the major cause of treatment failure. Recent estimates are that nearly 50% of drug-experienced patients in North America harbor HIV that is resistant to one or more of the 16 FDA-approved antiretroviral agents used in multi-drug ‘cocktails’ (Ref. dont have this ref). Moreover, it has been estimated that drug-resistant HIV accounts for up to 12% of new infections (Little et al., N. Engl. J. Med., 347:385 (2002)).

[0007] Accordingly, drug resistant HIV strains represent distinct infectious entities from a therapeutic viewpoint, and pose new challenges for drug design as well as drug treatment of existing infections. Substitutions have been documented in over 45 of the 99 amino acids of the HIV protease monomer in response to protease inhibitor treatment (Mellors, et al., International Antiviral News, 3:8 (1995); Eastman, et al., J. Virol., 72:5154 (1998); Kozal, et al., Nat. Med., 2:753 (1996)). The particular sequence and pattern of mutations selected by PIs is believed to be somewhat drug-specific and often patient-specific, but high level resistance is typified by multiple mutations in the protease gene which give rise to cross-resistance to all of the PIs.

[0008] In view of the foregoing problems, there exists a need for inhibitors against drug resistant and mdrHIV strains. Further, there exists a need for inhibitors against drug resistant and multi-drug resistant HIV proteases (mdrPR). Further still, there exists a need for inhibitors of HIV that can prevent or slow the emergence of drug resistant and mdrHIV strains in infected individuals.

[0009] Inhibitors with the ability to inhibit mdrHIV strains, and to slow the emergence of drug resistant strains in wild type HIV infections, are defined as “resistance-repellent” inhibitors.

[0010] There also exists a need for robust methods that can be used to design “resistance-repellent” inhibitors.

[0011] More generally, there is a need for therapeutic regimens that minimize the likelihood that resistance will occur in a disease involving a replicating biological entity. In one approach, drugs may be designed which have similar activity against both the wild type and mutant forms of their target. Such drugs minimize the probability of a mutant population emerging by reducing the selective pressure introduced by the drug when used to treat wild type infections. Such drugs also can be used to treat mutant infections and can be used for salvage therapy.

[0012] There is also an urgent need to develop potent, broad-spectrum, and mechanistically-novel antimicrobials suitable for tackling the growing problem of antibiotic-resistant bacteria strains, and for treating and/or preventing outbreaks of infectious diseases, including diseases caused by bioterrorism agents like anthrax, plague, cholera, gastroenteritis, multidrug-resistant tuberculosis (MDR TB). The recent anthrax attack of 2001 underscored the reality of large-scale aerosol bioweapons attack by terrorist groups. It also revealed that there is an urgent and pressing need to discover and develop novel and potent antimicrobials that can be used therapeutically and prophylactically for biodefense against new bioattacks. The NIH and CDC have identified a number of High Priority pathogens based on their likelihood of causing widespread contagious disease and/or death to the general population. Research on methods of protection against potential agents of bioterrorism has been a priority for several years at the NIH. A recent analysis suggested the existence of ongoing offensive biological weapons programs in at least 13 countries (Inglesby, T. V., et al., JAMA, 287:2236, (2002)).

[0013] The widespread use of antibiotics in human medical as well as in agricultural applications has promoted the emergence and spread of drug resistant bacteria that are no longer sensitive to existing drugs. The ease with which drug resistant microorganisms can be selected in a simple laboratory setting is a further concern when contemplating pharmaceutical-based strategies for biodefense. There is an urgent need to discover and develop novel therapeutic agents to combat pathogens that are likely to be used in a bioterrorist scenario.

[0014] A list of selected agents rated by likelihood to cause the greatest harm in a bioterrorist attack has been compiled by the CDC and NIAID (Lane, H. C., et al., Nat Med., 7:1271 (2001)). B. anthracis; the bacterium that causes anthrax, is one of the most serious of the group A pathogens. Dissemination of B. anthracis spores via the US Postal Service in 2001 established the feasibility of large-scale aerosol bioweapons attack. It has been estimated that between 130,000 and 3 million deaths would follow the release of 100 kg of B. anthracis, a lethality matching that of a hydrogen bomb (Inglesby, T. V., et al., JAMA, 287:2236, (2002)). Penicillin, doxycycline and ciprofloxacin are currently approved by the FDA for the treatment of inhalation anthrax infections. However, it was advised that antibiotic resistance to penicillin- and tetracycline-class antibiotics should be assumed following a terrorist attack (Inglesby, T. V., et al., JAMA, 281:1735-45 (1999)). Moreover, in vitro selection of a B. anthracis strain that is resistant to ofloxacin (a fluoroquinilone closely related to ciprofloxacin) has been reported (Choe, C. H., et al., Antimicrob. Agents. Chemother., 44:1766 (2000)). Following the anthrax attacks of 2001, the CDC advocated the use of a combination of 2-3 antibiotics. As a post-exposure prophylaxis, 60 days of treatment with ciprofloxacin is currently recommended. Strict compliance to this drug regimen is complicated by moderate to severe gastrointestinal tract intolerance.

[0015] Another group A pathogen, Y. pestis, is the causative agent of plague. If 50 kg of Y. pestis were released as an aerosol over a city of 5 million, pneumonic plague would afflict an estimated 150,000 individuals and result in 36,000 deaths (Inglesby, T. V., et al., JAMA, 283: 2281, (2000)). Streptomycin, tetracycline and doxycycline are the FDA-approved treatment for plague. Wide spread use of these antibiotics in the US raises concerns about possible resistance. A US-licensed, formaldehyde-killed whole bacilli vaccine was discontinued by its manufacturers in 1999 and is no longer available.

[0016] C. jejuni and V. cholerae are category B pathogens which can present a significant threat to the safety of food and water supplies. C. jejuni infections are one of the most commonly identified causes of acute bacterial gastroenteritis worldwide and area frequent cause of Traveler's diarrhea (Allos, B. M., Clin Infect Dis, 32:1201 (2001)). Currently, the CDC estimates that 2.4 million cases of Campylobacter infection occur in the United States each year, affecting almost 1% of the entire population. In the past few years, a rapidly increasing proportion of Campylobacter strains all over the world have been found to be fluoroquinolone-resistant. High rates of resistance make tetracycline, amoxicillin, ampicillin, metronidazole, and cephalasporins poor choices for the treatment of C. jejuni infections. All Campylobacter species are inherently resistant to vancomycin, rifampin, and trimethoprim. V. cholerae, a causative agent of cholera, is responsible for 120,000 deaths annually (Faruque, S. M., et al., Microbiol Mol Biol Rev, 62:1301 (1998)) and is characterized by a rapidly changing pattern of antibiotic resistance.

[0017] TB is one of the most common and devastating infectious diseases known to man. An estimated one third of the global population is infected with Mycobacteria tuberculosis. Eight million people develop an active infection and 2 million victims die yearly (Dye, C., et al., JAMA, 282:677 (1999.)). Currently, a combination of four drugs is recommended for TB treatment: isoniazid, rifampicin, pyrazinamide and ethambutole. The treatment course lasts 6 months. Such a multidrug combination together with the lengthy duration of treatment is prone to side-effects and adherence problems, which in turn can often lead to the development of drug resistance. The current drugs used to treat TB infections were introduced into clinical practice more than 30 years ago, in the absence of any knowledge of molecular mechanism. There is an urgent need to identify novel, effective, non-toxic and specific drugs that can shorten the duration of treatment, reduce side-effects, combat latent infection and reduce the spread of MDR TB strains. In addition, it is important to recognize the need for mechanistically novel drugs, i.e., antimicrobial agents that target biochemical pathways distinct from those of existing TB drugs, in order to be effective against MDR TB strains.

[0018] In summary, there is a clear need for the discovery of novel, non-toxic, broad spectrum antibiotics that can be used to (1) treat drug-resistant bacterial infections, and (2) protect civilians and military personnel in case of bioterrorist attacks. In one approach, drugs may be designed which have similar activity against both the wild type and variant forms of their target. Such drugs should minimize the probability of the emergence of mutant populations by reducing the selective pressure introduced by the drug when used to treat wild type infections. Such drugs also can be used to treat mutant infections and can be used for salvage therapy. In another approach, drugs may be designed which have similar activity against various isotypes of a homologous target. Such drugs can be used to treat multiple species of pathogenic microorganisms since they will be active against the target of each species. In a third approach, drugs can be designed that combine the properties and the uses of both of the above approaches.

[0019] There also exists a need for robust methods that can be used to design such broad spectrum antibiotics.

SUMMARY OF THE INVENTION

[0020] In a first aspect the invention features a method for the structure-based design of a drug that can act as an inhibitor of at least two different biological entities, the method comprising the steps of: (a) providing at least one structure of a wild type target protein or an inhibitor-wild type target protein complex; (b) providing at least one structure of a variant target protein or an inhibitor-variant target protein complex; (c) comparing at least one structure from step (a) with at least one structure from step (b) to determine whether there exists a common three-dimensionally conserved substructure comprising the atomic coordinates of the structurally conserved atoms of the inhibitors and structurally conserved atoms of the target proteins; and (d) if a conserved substructure exists, using the atomic coordinates of the conserved substructure to select a compound having atoms matching those of the structurally conserved atoms of the inhibitors, wherein the selection of the compound is performed using computer modeling.

[0021] The invention also features a method for the structure-based drug design of a broad spectrum compound, the method comprising the steps of: (a) providing at least one structure of a wild type target protein or an inhibitor-wild type target protein complex; (b) providing at least one structure of a variant target protein or an inhibitor-variant target protein complex; (c) comparing at least one structure from step (a) with at least one structure from step (b) to determine whether there exists a common three-dimensionally conserved substructure comprising the atomic coordinates of the structurally conserved atoms of the target proteins or a common three-dimensionally conserved substructure comprising the atomic coordinates of the structurally conserved atoms of the inhibitors and structurally conserved atoms of the target proteins; and (d) if a conserved substructure exists, using the atomic coordinates of the conserved substructure to select a compound having atoms matching those of the structurally conserved atoms of the inhibitors or to design a compound that binds to the target protein, wherein the selection of the compound is performed using computer modeling.

[0022] Desirably, the above method further comprises the steps of: (e) comparing at least one structure from step (a) with at least one structure from step (b) to determine whether there exists a three-dimensionally non-conserved substructure comprising the atomic coordinates of the structurally non-conserved atoms of the inhibitors and structurally non-conserved atoms of the target proteins; and (f) if a non-conserved substructure exists, using the atomic coordinates of the non-conserved substructure to reject a compound having atoms matching those of the structurally non-conserved atoms of the inhibitors, wherein the rejection of the compound is performed in conjunction with computer modeling.

[0023] In any of the above methods, at least two, four, six, or eight structures from step b can be used in step c. The methods can be applied using several structures, including at least two, four, six, or eight variant forms of the target protein.

[0024] The inhibitors used in the inhibitor-wild type target protein complex and the inhibitor-variant target protein complex can be the same or different. The inhibitors can be selected from competitive or noncompetitive inhibitors. Furthermore, the inhibitors can be selected from reversible, or irreversible inhibitors.

[0025] In any of the above methods, the variant target protein can be a homologous protein or a mutant protein.

[0026] In any of the above methods, the structures can be selected from crystal structures, NMR structures, computer models, any acceptable experimental, theoretical or computational method of deriving a three-dimensional representation of a structure, or a combination thereof.

[0027] Target proteins for use in the present invention include any therapeutically relevant protein. The target protein can be a viral, bacterial, protozoan, or fungal protein. In some instances, the target protein is one that is expressed in a neoplasm.

[0028] Preferably, the target protein can be an enzyme, a receptor, a structural protein, a component of a macromolecular complex, a component of a metabolic pathway, or an assembly of biological molecules. Desirably, the target protein is necessary for the survival of the replicating biological entity. For example, the target protein can be an enzyme selected from the group consisting of reverse transcriptases, proteases, DNA and RNA polymerases, methylases, oxidases, esterases, acyl transferases, helicases, topoisomerases, and kinases. The target protein can be a component of a metabolic pathway, such as the shikimate pathway. Desirable target proteins include HIV protease or 3-dehydroquinate dehydratase, among others.

[0029] Where the target protein is HIV protease, suitable inhibitors for use in the methods of the invention include those selected from the group consisting of indinavir, nelfinavir, ritonavir, saquinavir, amprenavir, lopinavir, and UIC-94003.

[0030] A broad spectrum protease inhibitor can be designed using the susbstructure of structurally conserved atoms described by the atomic coordinates in Table 8, which includes the structurally conserved atoms of the inhibitor and structurally conserved atoms of the protease. A broad spectrum protease inhibitor can also be designed using the structurally conserved atoms of the inhibitor alone. These are described by the atomic coordinates in Table 8.

[0031] A broad spectrum 3-dehydroquinate dehydratase inhibitor can be designed using the susbstructure of structurally conserved atoms described by the atomic coordinates in Table 12, which includes the structurally conserved atoms of the 3-dehydroquinate dehydratase. A broad spectrum 3-dehydroquinate dehydratase inhibitor can also be designed using the structurally conserved atoms of the inhibitor alone. These are described by the atomic coordinates in Table 12.

[0032] The invention also features compounds having a chemical structure selected using any of the methods above. Such compounds are broad spectrum inhibitors and have broad spectrum activity against replicating biological entities expressing a particular target protein. Thus, if the target protein is expressed by a microbe or a neoplasm, the compound will have broad spectrum activity against the microbe or neoplasm, respectively.

[0033] The invention features a compound having broad spectrum activity against HIV protease wherein the compound has a chemical structure selected using the methods above, including those methods utilizing the atomic coordinates of Table 8.

[0034] The invention features a compound having broad spectrum activity against 3-dehydroquinate dehydratase wherein the compound has a chemical structure selected using the methods above, including those methods utilizing the atomic coordinates of Table 12.

[0035] The compounds of the invention exclude bis-THF compounds (e.g., analogs of compounds 1 and 3) as described in J. Med. Chem. 39:3278-3290 (1996) (compounds 49-52 and 58-60), Bioorg. Med. Chem. Lett. 8:979-982 (1998), WO99/65870, U.S. Pat. No. 6,319,946, WO02/08657, WO02/092595, WO99/67417, EP00/9917, and WO00/76961; and also exclude fused ring THF structures as described in Bioorg. Med. Chem. Lett. 8:687-690 (1998) and U.S. Pat. No. 5,990,155.

[0036] For any of the broad spectrum inhibitors of the invention, broad spectrum activity can be measured by the ratio of the inhibitory concentrations of the broad spectrum inhibitor for the variant and wild type biological entities (IC50, variant/IC50, wild type). Desirably, the IC50, variant/IC50, wild type ratio for a broad spectrum inhibitor is less than 100, 80, 60, 40, 30, 20, 10, 8, 6, or, most desirably, less than 3.

[0037] A broad spectrum inhibitor can be active against several different mutant biological entities. Desirably, the inhibitor will have broad spectrum activity against at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mutant biological entities.

[0038] A broad spectrum inhibitor can also be active against different organisms or neoplastic cell types expressing homologous target proteins that possess sufficient structural similarity. Desirably, the inhibitor will have broad spectrum activity against at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 different organisms or neoplastic cell types expressing homologous target proteins.

[0039] The invention also features a pharmaceutical composition that includes a broad spectrum inhibitor described herein in any pharmaceutically acceptable form, including isomers such as diastereomers and enantiomers, salts, solvates, and polymorphs thereof. The composition can include an inhibitor of the invention along with a pharmaceutically acceptable carrier or diluent.

[0040] The invention also features methods of treating disease in a patient in need thereof, which includes the administration of a pharmaceutical composition of the invention to the patient in an amount sufficient to treat the disease. The pharmaceutical composition includes any broad spectrum inhibitor described herein. Such broad spectrum inhibitors have broad spectrum activity against replicating biological entities expressing a particular target protein. Thus, if the target protein is expressed by a microbe or a neoplasm, the disease to be treated will be a microbial infection or neoplasm, respectively.

[0041] The invention features a method of treating an HIV infection in a patient in need thereof, the method including the step of administering to the patient a pharmaceutical composition including a broad spectrum protease inhibitor described herein in amounts effective to treat the HIV infection.

[0042] The invention features a method of treating a bacterial infection in a patient in need thereof, the method including the step of administering to the patient a pharmaceutical composition including a broad spectrum 3-dehydroquinate dehydratase inhibitor described herein in amounts effective to treat the bacterial infection. The bacterial infection to be treated using the above method can be caused by a bacterium selected from the group consisting of C. jejuni, V. cholerae, Y. pestis, B. anthracis, P. putidas, and M. tuberculosis. Furthermore, this method can be used to treat infections by any microbe the utilizes 3-dehydroquinate dehydratase.

[0043] The invention also features the use of a pharmaceutical composition described herein in the manufacture of a medicament for the treatment of a disease. The pharmaceutical composition includes any broad spectrum inhibitor described herein. Such broad spectrum inhibitors and have broad spectrum activity against replicating biological entities expressing a particular target protein. Thus, if the target protein is HIV protease or 3-dehydroquinate dehydratase, the disease to be treated will be an HIV infection or bacterial infection, respectively.

[0044] The term “replicating biological entity” includes, for example, bacteria, fungi, yeasts, viruses, protozoa, prions and neoplasms

[0045] Neoplasms include, for example, carcinomas of the bladder, breast, colon, kidney, liver, lung, head and neck, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, or skin; a hematopoietic tumor of lymphoid lineage; a hematopoietic tumor of myeloid lineage; a tumor of mesenchymal origin; a tumor of the central or peripheral nervous system; melanoma; seminoma; teratocarcinoma; osteosarcoma; thyroid follicular cancer; and Kaposi's sarcoma. Hematopoietic tumors of lymphoid lineage can be leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma.

[0046] By “wild type target protein” is meant a protein obtained from a replicating biological entity that has not been subjected to drug selection pressure, and could include polymorphisms or isoforms thereof. A replicating biological entity that expresses wild type target protein is referred to herein as a wild type biological entity.

[0047] By “variant target protein” is meant a mutant target protein or a homologous target protein. A replicating biological entity that expresses variant target protein is referred to herein as a variant biological entity.

[0048] By “mutant target protein” is meant a target protein that contains one or more amino acid substitutions with respect to the wild type target protein, including proteins from the same organism that have evolved under drug selection pressure. In general, mutant target proteins will have one or more amino acid substitutions and should be readily identified as related to the cognate wild type protein using standard sequence comparison methods. A replicating biological entity that expresses mutant target protein is referred to herein as a mutant biological entity.

[0049] By “homologous target protein” is meant a variant target protein that is expressed in a different species or neoplastic cell type than the wild type target protein, but has the same, or similar, function.

[0050] By “structurally conserved target substructure”, and by “structurally” or “three-dimensionally conserved substructure” as applied to target proteins, is meant the regions of the target protein structure which are not significantly affected by amino acid mutations or substitutions. Such regions can be defined using standard methods of comparative analysis of three-dimensional structures of proteins, such as superposition analysis, for example. In the case of HIV protease, these regions were identified using a pair wise superposition analysis of wild type and mutant protease structures complexed with inhibitors. The superposition of structures can be performed using the iterative procedure described herein. In the case of DHQase, these regions were identified using a pair wise superposition analysis of wild type and homologous DHQase structures from different bacterial species with and without inhibitors. It is apparent that the overall compositions of structurally conserved target substructures will likely differ for different, non-homologous target proteins, especially when the frequency of amino acid substitutions in high. However, a quantitative definition can be derived from the superposition analysis, which provides both the identities and the positions of the atoms that comprise these substructures. The regions that comprise structurally conserved target substructures contain atoms whose superimposed pairs have three-dimensional atomic coordinates that match to within a distance of 1 Å, 0.6 Å, 0.4 Å, or 0.2 Å.

[0051] By “broad spectrum inhibitor” is meant a compound having broad spectrum activity, i.e., an inhibitor that is active against two different-biological entities, e.g., both a wild type biological entity and one or more variants of that biological entity. Thus, broad spectrum activity can be described by the inhibitor's action against a particular target protein (e.g., broad spectrum activity against protease) or a particular target organism (e.g., broad spectrum activity against HIV). Broad spectrum inhibitors will have medically insignificant interactions with non-conserved regions. Broad spectrum inhibitors can be useful for the treatment and/or prevention of infectious diseases caused by multiple infectious agents, as well as for decreasing the development of drug-resistance by these organisms.

[0052] As used herein, the term “treating” refers to administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. To “prevent disease” refers to prophylactic treatment of a patient who is not yet ill, but who is susceptible to, or otherwise at risk of, a particular disease. To “treat disease” or use for “therapeutic treatment” refers to administering treatment to a patient already suffering from a disease to ameliorate the disease and improve the patient's condition. Thus, in the claims and embodiments, treating is the administration to a patient either for therapeutic or prophylactic purposes.

[0053] The term “microbial infection” refers to the invasion of the host patient by pathogenic microbes (e.g., bacteria, fungi, yeasts, viruses, protozoa). This includes the excessive growth of microbes that are normally present in or on the body of a patient. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host patient. Thus, a patient is “suffering” from a microbial infection when excessive numbers of a microbial population are present in or on a patient's body, or when-the presence of a microbial population(s) is damaging the cells or other tissue of a patient.

[0054] The term “microbes” includes, for example, bacteria, fungi, yeasts, viruses and protozoa. The term “administration” or “administering” refers to a method of giving a dosage of a pharmaceutical composition to a patient, where the method is, e.g., topical, oral, intravenous, intraperitoneal, or intramuscular. The preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, site of the potential or actual disease and severity of disease.

[0055] The term “patient” includes humans, cattle, pigs, sheep, horses, dogs, and cats, and also includes other vertebrate, most preferably, mammalian species.

[0056] Where “atomic coordinates” are provided, or otherwise referred to, these coordinates define a three dimensional structure. That such a structure may be defined by more than one different coordinate system, e.g., by translation or rotation of the coordinates, does not change the relative positions of the atoms in the structure. Accordingly, any reference to atomic coordinates herein is intended to include any equivalent three dimensional structure defined by the coordinates.

[0057] By “computer modeling” is meant the use of a computer to visualize or compute a compound, a portion of a compound, a target protein, a portion of a target protein, a complex between a compound and a target protein, or a portion of a complex between a compound and a target protein.

[0058] Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG. 1 is a table depicting the structures of compounds 1-7, gt33, and qxa.

[0060] FIG. 2 illustrates the amino acid alignment of type II DHQases. Fully conserved residues are framed. Catalytically important amino acids are marked by stars. Arrows denote amino acids that make hydrogen bonds and ionic interactions in the structure of M. tuberculosis DHQase complexed with the inhibitor, 3-dehydroquinic acid oxime.

[0061] FIG. 3 illustrates the key interactions of the substrate-based inhibitor, DHQO, with the active site residues for the Type II DHQase from M. tuberculosis.

DETAILED DESCRIPTION

[0062] We have discovered that the comparative analysis of the structures of complexes of inhibitors bound to wild type and variant forms of a target protein can be used to design compounds that are broad spectrum inhibitors.

[0063] The methods of the invention entail the design of compounds having a particular structure. The methods rely upon the use of structural information to arrive at these compounds. The structural data define a three dimensional array-of the important contact atoms in an inhibitor that bind to the target protein in a fashion that results in broad spectrum activity against biological entities expressing variants of the target protein.

[0064] Inhibitor-Target Protein Structures

[0065] Atomic structural coordinates can be selected from crystal structures, NMR structures, computer models, any acceptable experimental, theoretical or computational method of deriving a three-dimensional representation of a structure, or a combination thereof. Atomic coordinates for use in the methods of the invention can be obtained from publicly available sources, e.g. from the Protein Data Bank, or obtained using known experimental or computational methods.

[0066] Atomic structural coordinates for use in the methods of the invention include crystal structures of HIV protease/inhibitor complexes derived from wild type and drug-resistant mutant proteases, and of DHQase and DHQase inhibitor complexes derived from two or more bacterial species, among others. In examples 1-3, the methods of the invention are applied using the coordinates of wild type HIV protease complexed with amprenavir, wild type HIV protease complexed with UIC-94003, and V82F/184V mutant HIV protease complexed with UIC-94003. In example 4, the methods of the invention are applied using the coordinates of wild type DHQase from M. tuberculosis and from Pseudomonas putidas. a complex between a compound and a target protein. The coordinates of other representative structures of HIV protease and DHQase should be useful for performing the methods of the present invention.

[0067] Conserved Substructures

[0068] Conserved substructures can be identified for target proteins, for target protein-inhibitor complexes, and/or for inhibitors, depending on the nature of the structures that are used in the comparative superposition analysis. In one approach, at least one structure of a wild type target protein is compared to at least one structure of a mutant or homologous target protein to determine whether a common three-dimensionally conserved substructure is present among the wild type protein and the mutant or homologous proteins, respectively. In another approach, at least one structure of an inhibitor-wild type target protein complex and at least one structure of an inhibitor-mutant target protein complex are compared to determine whether a common three-dimensionally conserved substructure is present among the mutant and wild type complexes. In a third approach, at least one structure of an inhibitor-wild type target protein complex and at least one structure of a mutant or homologous target protein without inhibitor are compared to determine whether a common three-dimensionally conserved substructure is present among the respective mutant or homologous protein and the wild type complexes. Variations of the approached described above can also be used. In each case, such a comparison can be made by means of (a) an overall superposition of the atoms of the protein structures; and, where feasible, (b) a study of the distances from atoms of the inhibitors to atoms of the protein. This analysis requires three-dimensional atomic coordinates of the protein structures and of the bound inhibitor.

[0069] The superposition of the protein structures can be performed in a two step process: 1) the distance between all pairs of corresponding Cá atoms (Cá atom of residue number 1 in one protein to Cá atom of residue number 1 in the second protein; Cá atom of residue number 2 in one protein to Cá atom of residue number 2 in the second protein; and so on) of the polypeptide chains is minimized by means of a least-square algorithm; 2) the superposition is refined by minimizing, in an iterative process, the distances between corresponding Cá atoms that are closer than a given distance (0.25 A for example), thus eliminating regions of the structures having large conformational differences to compute the superposition parameters. Furthermore, where a partial structure is provided (e.g., from NMR data) the available coordinates are superimposed.

[0070] The conserved substructure identifies the relevant portion of the target protein that is the active site, or binding region, defined by that part of the target protein interacting with inhibitor. Important interactions between the target protein and inhibitor are identified by mapping the contacts between the two. Structurally conserved regions of the target protein not near the binding site are generally not relevant to the design of the broad spectrum inhibitor. Accordingly, the selection of the meaningful substructure is identified using the above mentioned contacts.

[0071] Design of a Broad Spectrum Inhibitor

[0072] The coordinates of the conserved inhibitor substructure are used to design an inhibitor having atoms matching those of the three-dimensionally structurally conserved atoms of the inhibitors. The result is an inhibitor for which IC50, variant and IC50, wild type are similar, minimizing the selective pressure introduced by the drug.

[0073] The methods of the invention can employ computer-based methods for designing broad spectrum inhibitors. These computer-based methods fall into two broad classes: database methods and de novo design methods. In database methods the compound of interest is compared to all compounds present in a database of chemical structures and compounds whose structure is in some way similar to the compound of interest are identified. The structures in the database are based on either experimental data, generated by NMR or x-ray crystallography, or modeled three-dimensional structures based on two-dimensional (i.e., sequence) data. In de novo design methods, models of compounds whose structure is in some way similar to the compound of interest are generated by a computer program using information derived from known structures, e.g., data generated by x-ray crystallography and/or theoretical rules. Such design methods can build a compound having a desired structure in either an atom-by-atom manner or by assembling stored small molecular fragments.

[0074] The success of both database and de novo methods in identifying compounds having the desired activity depends on the identification of the functionally relevant portion of the compound of interest. The functionally relevant portion of the compound, the pharmacophore, is defined by the structurally conserved substructure. A pharmacophore then is an arrangement of structural features and functional groups important for obtaining an inhibitor having broad spectrum activity.

[0075] Not all identified compounds having the desired pharmacophore will act as broad spectrum inhibitors. The actual activity can be finally determined only by measuring the activity of the compound in relevant biological assays. However, the methods of the invention are extremely valuable because they can be used to greatly reduce the number of compounds which must be tested to identify those likely to exhibit broad spectrum activity.

[0076] Programs suitable for generating predicted three-dimensional structures from two-dimensional data include: Concord (Tripos Associated, St. Louis, Mo.), 3-D Builder (Chemical Design Ltd., Oxford, U.K.), Catalyst (Bio-CAD Corp., Mountain View, Calif.), and Daylight (Abbott Laboratories, Abbott Park, Ill.).

[0077] Programs suitable for searching three-dimensional databases to identify molecules bearing a desired pharmacophore include: MACCS-3D and ISIS/3D (Molecular Design Ltd., San Leandro, Calif.), ChemDBS-3D (Chemical Design Ltd., Oxford, U.K.), and Sybyl/3DB Unity (Tripos Associates, St. Louis, Mo.).

[0078] Programs suitable for pharmacophore selection and design include: DISCO (Abbott Laboratories, Abbott Park, Ill.), Catalyst (Bio-CAD Corp., Mountain View, Calif.), and ChemDBS-3D (Chemical Design Ltd., Oxford, U.K.).

[0079] Databases of chemical structures are available from Cambridge Crystallographic Data Centre (Cambridge, U.K.) and Chemical Abstracts Service (Columbus, Ohio).

[0080] De novo design programs include Ludi (Biosym Technologies Inc., San Diego, Calif.) and Aladdin (Daylight Chemical Information Systems, Irvine Calif.).

[0081] One skilled in the art may use one of several methods to screen chemical entities for their ability to match the conserved substructure. This process may begin by visual inspection of, for example, the active site on the computer screen based on the atomic coordinates for the target protein. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.

[0082] Specialized computer programs may also assist in the process of selecting chemical entities. These include:

[0083] 1. GRID (Goodford, P. J., “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules,” J. Med. Chem., 28:849 (1985)). GRID is available from Oxford University, Oxford, UK.

[0084] 2. MCSS (Miranker, A. and M. Karplus, “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins: Structure, Function, and Genetics, 11:29 (1991)). MCSS is available from Molecular Simulations, Burlington, Mass.

[0085] 3. AUTODOCK (Goodsell, D. S. and A. J. Olsen, “Automated Docking of Substrates to Proteins by Simulated Annealing,” Proteins: Structure, Function, and Genetics, 8:195 (1990)). AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.

[0086] 4. DOCK (Kuntz, L. D. et al., “A Geometric Approach to Macromolecule-Ligand Interactions,” J. Mol. Biol., 161:269 (1982)). DOCK is available from University of California, San Francisco, Calif.

[0087] Once the conserved substructure for the inhibitor has been identified, the conserved atoms of the inhibitor can be selected for assembly into a single inhibitor. Assembly may be proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of the target protein. This may be followed by manual model building using software such as Quanta or Sybyl.

[0088] Useful programs to aid one of skill in the art in assembly of the individual chemical entities or fragments include:

[0089] 1. CAVEAT (Bartlett, P. A. et al, “CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules”. In “Molecular Recognition in Chemical and Biological Problems,” Special Pub., Royal Chem. Soc., 78:182 (1989)). CAVEAT is available from the University of Calif., Berkeley, Calif.

[0090] 2. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Martin, Y. C., “3D Database Searching in Drug Design,” J. Med. Chem., 35:2145 (1992)).

[0091] 3. HOOK (available from Molecular Simulations, Burlington, Mass.).

[0092] Other molecular modeling techniques may also be employed in accordance with this invention. See, e.g., Cohen, N. C. et al., “Molecular Modeling Software and Methods for Medicinal Chemistry,” J. Med. Chem., 33:883 (1990). See also, Navia, M. A. and M. A. Murcko, “The Use of Structural Information in Drug Design,” Current Opinions in Structural Biology, 2:202 (1992).

[0093] Once a broad spectrum inhibitor has been optimally designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided.

[0094] In general, inhibitors designed using the methods of the invention can be tested for broad spectrum activity using any of the to in vitro and/or in vivo methods described below, among others.

[0095] Broad Spectrum Inhibitors

[0096] Broad spectrum inhibitors match the pharmacophore defined by the structurally conserved substructure. The pharmacophore is the arrangement of structural features and functional groups important for obtaining an inhibitor having broad spectrum activity. This pharmacophore is derived using structural data for known inhibitors complexed to a target protein. Accordingly, broad spectrum inhibitors will often be structurally related to known compounds lacking broad spectrum activity, but useful in the design of broad spectrum inhibitors using the methods disclosed herein. These known inhibitors serve as lead compounds for both the design and synthesis of a broad spectrum inhibitor. Using the synthetic methods for making the lead compounds and standard synthetic methods as described by, for example, J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure,” John Wiley & Sons, Inc., 1992; T. W. Green and P. G. M. Wuts, “Protective Groups in Organic Synthesis” (2nd Ed.), John Wiley & Sons, 1991; and P. J. Kocienski, “Protecting Groups,” Georg Thieme Verlag, 1994, one can synthesize the broad spectrum inhibitors described herein.

[0097] Typically the lead compounds bear varied functional groups which are present in the pharmacophore, including hydrogen-bond donors, hydrogen-bond acceptors, ionic moieties, polar moieties, hydrophobic moieties, aromatic centers, and electron-donors and acceptors. These are linked by a structural scaffold which imparts the appropriate a three dimension arrangement of the functional groups.

[0098] Numerous modifications of the lead compound can be made using techniques known in the art. These include changing a functional group by replacing it with another moiety of the same group. For example, one hydrogen-bond donor may be substituted by another. A good hydrogen bond donor has an H atom bonded to a very electronegative atom (e.g., O—H or N—H). Examples of hydrogen-bond donors include alcohols, carboxylic acids, oximes, and amides, among others. Similarly, one hydrogen-bond acceptor may be substituted by another. A good hydrogen bond acceptor has an electronegative element with lone pairs (e.g., O, N, or F). Examples of hydrogen bond acceptors include water, halogen atoms, alcohols, amines, carbonyls, ethers, and amides, among others. It may also be desirable to alter the distance between functional groups in a lead compound. This is achieved by employing synthetic methods analogous to those used to prepare the lead compound, but replacing the scaffold with a structurally related scaffold that provides the desired distance (e.g., a scaffold that incorporates more or fewer atoms linking the relevant functional groups). In some instances it may also be desirable to alter the stereochemistry in a lead compound. This can be accomplished by employing racemic starting materials, or by employing reaction conditions that result in racemization of the relevant chiral center, followed by separation of the enantiomeric or diastereomeric mixture.

[0099] Assays

[0100] Inhibitors designed using the methods disclosed herein may be further assayed, using standard in vitro models or animal models, to evaluate therapeutic activity and toxicity. These assays are described in the literature and are familiar to those skilled in the art. These include but are not limited to assays for monitoring or measuring efficacy against HIV, bacteria, and neoplasms.

[0101] One skilled in the art will be familiar with methods of measuring the IC50's of a broad spectrum inhibitor described herein. The IC50 value is determined by plotting percent activity versus inhibitor concentration in the assay and identifying the concentration at which 50% of the activity (e.g., growth, enzymatic activity, protein production, etc.) remains. Inhibitors can be tested for antimicrobial activity against a panel of organisms according to standard procedures described by the National Committee for Clinical Laboratory Standards (NCCLS document 7-A3, Vol. 13, No. 25, 1993/NCCLS document M27-P, Vol. 12, No. 25, 1992). Inhibitors can be dissolved (0.1 μg/ml-500 μg/ml) in microbial growth media, diluted, and added to wells of a microtiter plate containing bacteria or fungal cells in a final volume of an appropriate media (Mueller-Hinton Broth; Haemophilus Test Media; Mueller-Hinton Broth+5% Sheep Blood; or RPMI 1690). Typically, the plates are incubated overnight at an appropriate temperature (30° C. to 37° C.) and optical densities (measure of cell growth) are measured using a commercial plate reader.

[0102] IC50 's for broad spectrum protease inhibitors can be measured against wild type HIV and clinically isolated mutant HIV isolates, utilizing the PHA-PBMC exposed to HIV-1 (50 TCID50 dose/X06 PBMC) as target cells and using the inhibition of p24 Gag protein production as an endpoint. The amounts of p24 antigen produced by the cells can be determined on day 7 in culture using a commercially available radioimmunoassay kit. Drug concentrations resulting in 50% inhibition (IC50's) of p24 antigen production can be determined by comparison with the p24 production level in drug-free control cell cultures.

[0103] Therapy

[0104] The invention features a method of identifying a compound having broad spectrum activity. Broad spectrum inhibitors of the present invention may be administered by any appropriate route for treatment or prevention of a disease or condition associated with a bacterial infection, viral infection, or neoplastic disorder, among others. These may be administered to humans, domestic pets, livestock, or other animals with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Administration may be topical, parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracistemal, intraperitoneal, intranasal, aerosol, by suppositories, or oral administration.

[0105] Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

[0106] Methods well known in the art for making formulations are found, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro AR., 2000, Lippincott Williams & Wilkins). Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. The concentration of the broad spectrum inhibitor in the formulation will vary depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.

[0107] The broad spectrum inhibitor may be optionally administered as a pharmaceutically acceptable salt, such as a non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and calcium, among others.

[0108] Administration of compounds in controlled release formulations is useful where the broad spectrum inhibitor has (i) a narrow therapeutic index (e.g., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; generally, the therapeutic index, TI, is defined as the ratio of median lethal dose (LD50) to median effective dose (ED50)); (ii) a narrow absorption window in the gastro-intestinal tract; or (iii) a short biological half-life, so that frequent dosing during a day is required in order to sustain the plasma level at a therapeutic level.

[0109] Many strategies can be pursued to obtain controlled release in which the rate of release outweighs the rate of metabolism of the broad spectrum inhibitor. For example, controlled release can be obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.

[0110] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).

[0111] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.

[0112] Pharmaceutical formulations of broad spectrum inhibitor described herein include isomers such as diastereomers and enantiomers, mixtures of isomers, including racemic mixtures, salts, solvates, and polymorphs thereof.

[0113] The formulations can be administered to human patients in therapeutically effective amounts. For example, when the broad spectrum inhibitor is an antimicrobial drug, an amount is administered which prevents, stabilizes, eliminates, or reduces a microbial infection. Typical dose ranges are from about 0.01 μg/kg to about 2 mg/kg of body weight per day. The exemplary dosage of drug to be administered is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipients, and its route of administration. Standard clinical trials maybe used to optimize the dose and dosing frequency for any particular broad spectrum inhibitor.

[0114] The following examples are meant to illustrate, but in no way limit, the claimed invention.

EXAMPLE I

[0115] This example illustrates the method by which experimentally-determined crystal structures of the same inhibitor in complex with wild type and mutant species of HIV protease can be compared and analyzed for the existence of a three-dimensionally conserved substructure.

[0116] The structures of wild type HIV-1 protease and a mutant, V82F/184V, HIV-1 protease, both in complexes with the inhibitor shown in FIG. 1 were determined using conventional x-ray crystallography techniques. The structures were analyzed by means of (a) an overall superposition of the atoms of the protein structures; and, (b) a study of the distances from atoms of the inhibitors to atoms of the protein. This analysis requires three dimensional atomic coordinates of the protein structures and of the bound inhibitor.

[0117] The superposition of the protein structures was performed in a two step process: 1) the distance between all pairs of corresponding Cá atoms (Cá atom of residue number 1 in one protein to Cá atom of residue number 1 in the second protein; Cá atom of residue number 2 in one protein to Cá atom of residue number 2 in the second protein; and so on) of the polypeptide chains is minimized by means of a least-square algorithm; 2) the superposition is refined by minimizing, in an iterative process, the distances between corresponding Cá atoms that are closer than a given distance (0.25 Å in this example), thus eliminating regions of the structures having large conformational differences to compute the superposition parameters. The distances between equivalenced Cá atoms after the minimization procedure are shown in Table 4.

TABLE 4
Distances between equivalent Cá atoms
Molecule 1: HIV-1 PR wt: 1
Molecule 2: HIV-1 PR V82F/I84V mutant: 1
	Molecule 1	Molecule 2	distance [Å]
	CA PRO 1	CA PRO 1	0.455
	CA GLN 2	CA GLN 2	0.434
	CA ILE 3	CA ILE 3	0.418
	CA THR 4	CA THR 4	0.317
	CA LEU 5	CA LEU 5	0.172
	CA TRP 6	CA TRP 6	0.228
	CA GLN 7	CA GLN 7	0.364
	CA ARG 8	CA ARG 8	0.166
	CA PRO 9	CA PRO 9	0.057
	CA LEU 10	CA LEU 10	0.183
	CA VAL 11	CA VAL 11	0.194
	CA THR 12	CA THR 12	0.168
	CA ILE 13	CA ILE 13	0.146
	CA LYS 14	CA LYS 14	0.229
	CA ILE 15	CA ILE 15	0.266
	CA GLY 16	CA GLY 16	0.662
	CA GLY 17	CA GLY 17	0.491
	CA GLN 18	CA GLN 18	0.267
	CA LEU 19	CA LEU 19	0.112
	CA LYS 20	CA LYS 20	0.128
	CA GLU 21	CA GLU 21	0.190
	CA ALA 22	CA ALA 22	0.169
	CA LEU 23	CA LEU 23	0.218
	CA LEU 24	CA LEU 24	0.233
	CA ASP 25	CA ASP 25	0.160
	CA THR 26	CA THR 26	0.200
	CA GLY 27	CA GLY 27	0.303
	CA ALA 28	CA ALA 28	0.169
	CA ASP 29	CA ASP 29	0.150
	CA ASP 30	CA ASP 30	0.038
	CA THR 31	CA THR 31	0.047
	CA VAL 32	CA VAL 32	0.173
	CA LEU 33	CA LEU 33	0.194
	CA GLU 34	CA GLU 34	0.310
	CA GLU 35	CA GLU 35	0.260
	CA MET 36	CA MET 36	0.136
	CA SER 37	CA SER 37	0.494
	CA LEU 38	CA LEU 38	0.607
	CA PRO 39	CA PRO 39	0.094
	CA GLY 40	CA GLY 40	0.774
	CA ARG 41	CA ARG 41	0.448
	CA TRP 42	CA TRP 42	0.204
	CA LYS 43	CA LYS 43	0.596
	CA PRO 44	CA PRO 44	0.625
	CA LYS 45	CA LYS 45	0.541
	CA MET 46	CA MET 46	0.643
	CA ILE 47	CA ILE 47	0.361
	CA GLY 48	CA GLY 48	0.240
	CA GLY 49	CA GLY 49	0.182
	CA ILE 50	CA ILE 50	0.110
	CA GLY 51	CA GLY 51	0.243
	CA GLY 52	CA GLY 52	0.200
	CA PHE 53	CA PHE 53	0.119
	CA ILE 54	CA ILE 54	0.255
	CA LYS 55	CA LYS 55	0.295
	CA VAL 56	CA VAL 56	0.108
	CA ARG 57	CA ARG 57	0.129
	CA GLN 58	CA GLN 58	0.074
	CA TYR 59	CA TYR 59	0.372
	CA ASP 60	CA ASP 60	0.496
	CA GLN 61	CA GLN 61	0.780
	CA ILE 62	CA ILE 62	0.406
	CA LEU 63	CA LEU 63	0.211
	CA ILE 64	CA ILE 64	0.260
	CA GLU 65	CA GLU 65	0.193
	CA ILE 66	CA ILE 66	0.181
	CA CYS 67	CA CYS 67	0.518
	CA GLY 68	CA GLY 68	0.641
	CA HIS 69	CA HIS 69	0.319
	CA LYS 70	CA LYS 70	0.179
	CA ALA 71	CA ALA 71	0.265
	CA ILE 72	CA ILE 72	0.350
	CA GLY 73	CA GLY 73	0.253
	CA THR 74	CA THR 74	0.301
	CA VAL 75	CA VAL 75	0.187
	CA LEU 76	CA LEU 76	0.186
	CA VAL 77	CA VAL 77	0.070
	CA GLY 78	CA GLY 78	0.306
	CA PRO 79	CA PRO 79	0.047
	CA THR 80	CA THR 80	0.470
	CA PRO 81	CA PRO 81	0.404
	CA VAL 82	CA PHE 82	0.556
	CA ASN 83	CA ASN 83	0.146
	CA ILE 84	CA VAL 84	0.196
	CA ILE 85	CA ILE 85	0.163
	CA GLY 86	CA GLY 86	0.224
	CA ARG 87	CA ARG 87	0.127
	CA ASN 88	CA ASN 88	0.048
	CA LEU 89	CA LEU 89	0.081
	CA LEU 90	CA LEU 90	0.197
	CA THR 91	CA THR 91	0.226
	CA GLN 92	CA GLN 92	0.176
	CA ILE 93	CA ILE 93	0.151
	CA GLY 94	CA GLY 94	0.338
	CA CYS 95	CA CYS 95	0.233
	CA THR 96	CA THR 96	0.305
	CA LEU 97	CA LEU 97	0.089
	CA ASN 98	CA ASN 98	0.260
	CA PHE 99	CA PHE 99	0.250
	CA PRO 101	CA PRO 101	0.227
	CA GLN 102	CA GLN 102	0.108
	CA ILE 103	CA ILE 103	0.206
	CA THR 104	CA THR 104	0.169
	CA LEU 105	CA LEU 105	0.125
	CA TRP 106	CA TRP 106	0.363
	CA GLN 107	CA GLN 107	0.296
	CA ARG 108	CA ARG 108	0.400
	CA PRO 109	CA PRO 109	0.173
	CA LEU 110	CA LEU 110	0.182
	CA VAL 111	CA VAL 111	0.085
	CA THR 112	CA THR 112	0.123
	CA ILE 113	CA ILE 113	0.107
	CA LYS 114	CA LYS 114	0.368
	CA ILE 115	CA ILE 115	0.226
	CA GLY 116	CA GLY 116	0.638
	CA GLY 117	CA GLY 117	0.516
	CA GLN 118	CA GLN 118	0.414
	CA LEU 119	CA LEU 119	0.102
	CA LYS 120	CA LYS 120	0.191
	CA GLU 121	CA GLU 121	0.206
	CA ALA 122	CA ALA 122	0.197
	CA LEU 123	CA LEU 123	0.231
	CA LEU 124	CA LEU 124	0.145
	CA ASP 125	CA ASP 125	0.235
	CA THR 126	CA THR 126	0.311
	CA GLY 127	CA GLY 127	0.200
	CA ALA 128	CA ALA 128	0.102
	CA ASP 129	CA ASP 129	0.143
	CA ASP 130	CA ASP 130	0.261
	CA THR 131	CA THR 131	0.172
	CA VAL 132	CA VAL 132	0.232
	CA LEU 133	CA LEU 133	0.103
	CA GLU 134	CA GLU 134	0.175
	CA GLU 135	CA GLU 135	0.190
	CA MET 136	CA MET 136	0.220
	CA SER 137	CA SER 137	0.739
	CA LEU 138	CA LEU 138	0.277
	CA PRO 139	CA PRO 139	0.325
	CA GLY 140	CA GLY 140	0.390
	CA ARG 141	CA ARG 141	0.174
	CA TRP 142	CA TRP 142	0.168
	CA LYS 143	CA LYS 143	0.304
	CA PRO 144	CA PRO 144	0.194
	CA LYS 145	CA LYS 145	0.456
	CA MET 146	CA MET 146	0.362
	CA ILE 147	CA ILE 147	0.178
	CA GLY 148	CA GLY 148	0.390
	CA GLY 149	CA GLY 149	0.434
	CA ILE 150	CA ILE 150	0.050
	CA GLY 151	CA GLY 151	0.199
	CA GLY 152	CA GLY 152	0.152
	CA PHE 153	CA PHE 153	0.455
	CA ILE 154	CA ILE 154	0.198
	CA LYS 155	CA LYS 155	0.470
	CA VAL 156	CA VAL 156	0.590
	CA ARG 157	CA ARG 157	0.607
	CA GLN 158	CA GLN 158	0.465
	CA TYR 159	CA TYR 159	0.301
	CA ASP 160	CA ASP 160	0.294
	CA GLN 161	CA GLN 161	0.308
	CA ILE 162	CA ILE 162	0.274
	CA LEU 163	CA LEU 163	0.235
	CA ILE 164	CA ILE 164	0.367
	CA GLU 165	CA GLU 165	0.410
	CA ILE 166	CA ILE 166	0.201
	CA CYS 167	CA CYS 167	0.409
	CA GLY 168	CA GLY 168	0.406
	CA HIS 169	CA HIS 169	0.410
	CA LYS 170	CA LYS 170	0.282
	CA ALA 171	CA ALA 171	0.273
	CA ILE 172	CA ILE 172	0.317
	CA GLY 173	CA GLY 173	0.563
	CA THR 174	CA THR 174	0.129
	CA VAL 175	CA VAL 175	0.237
	CA LEU 176	CA LEU 176	0.155
	CA VAL 177	CA VAL 177	0.240
	CA GLY 178	CA GLY 178	0.386
	CA PRO 179	CA PRO 179	0.340
	CA THR 180	CA THR 180	0.335
	CA PRO 181	CA PRO 181	0.446
	CA VAL 182	CA PHE 182	0.343
	CA ASN 183	CA ASN 183	0.205
	CA ILE 184	CA VAL 184	0.262
	CA ILE 185	CA ILE 185	0.096
	CA GLY 186	CA GLY 186	0.118
	CA ARG 187	CA ARG 187	0.202
	CA ASN 188	CA ASN 188	0.073
	CA LEU 189	CA LEU 189	0.108
	CA LEU 190	CA LEU 190	0.127
	CA THR 191	CA THR 191	0.177
	CA GLN 192	CA GLN 192	0.175
	CA ILE 193	CA ILE 193	0.241
	CA GLY 194	CA GLY 194	0.118
	CA CYS 195	CA CYS 195	0.375
	CA THR 196	CA THR 196	0.437
	CA LEU 197	CA LEU 197	0.167
	CA ASN 198	CA ASN 198	0.178

[0118] Table 4 shows that the 184V, V82F mutations induce structural changes relative to the wild type structure in some parts of the enzyme, but that other regions are less affected. The regions of the protein structure which are not significantly affected by the amino acid mutations are defined as structurally conserved regions. In the present example, the mutations result in localized structural changes in the backbone of HIV protease over a wide range, from 0.038-0.774 Å.

[0119] The distances between the strongly interacting atoms of the inhibitor to atoms of the wild type and mutant protein, that is hydrogen-bond donors and acceptors, were computed and they are displayed in Table 5.

TABLE 5
Distances between atoms of the inhibitor and atoms of
the protein
	HIV PR wt: 1	V82F/I84V: 1
	O2-Wat301	2.92	2.89
	N1-027	3.36	3.46
	O6-N30	3.30	3.61
	06-N29	3.19	3.55
	O7-N29	2.84	2.87
	O7-OD1 29	3.42	3.54
	O7-O1	3.31	3.19
	O3-OD 25 (out)	2.50	2.94
	O3-OD 25 (in)	2.65	2.67
	O3-OD125 (out)	3.27	3.21
	O3-OD125 (in)	2.80	2.67
	O5-Wat301	2.70	2.79
	O8-N130	3.16	2.96

[0120] Table 5 shows that the atoms of the inhibitor interact with the same atoms of the two different proteins, in this case the wild type and V82F/184V mutant HIV proteases. From Table 5, it can be seen that the atoms of the enzymes with which the inhibitor interacts belong to the structurally conserved regions. The effects of mutations on the protein-inhibitor interactions can be quantified in terms of the distances between interacting pairs of atoms from the inhibitor and from atoms of the three-dimensionally conserved substructure of the protein. These distances are similar in the wild type and in the mutant complexes; the average of their differences is only 0.07 Å. The range of the differences is 0.02-0.36 Å.

EXAMPLE 2

[0121] This example illustrates the method by which experimentally-determined crystal structures of two different inhibitors in complexes with wild type HIV protease can be compared and analyzed for the existence of a three-dimensionally conserved substructure. The structures of wild type HIV-1 protease in complexes with inhibitor 1 and with Amprenavir (inhibitor 2) were analyzed by means of (a) an overall superposition of the protein structures; and (b) a study of the distances from atoms of the inhibitors to atoms of the protein.

[0122] The superposition of the protein structures is performed in a two step process: 1) the distance between all pairs of corresponding Cá atoms (Cá atom of residue number 1 in one protein to Cá atom of residue number 1 in the second protein; Cá atom of residue number 2 in one protein to Cá atom of residue number 2 in the second protein; and so on) of the polypeptide chains is minimized by means of a least-square algorithm; 2) the superposition is refined by minimizing, in an iterative process, the distances between corresponding Cá atoms that are closer than a given distance (0.25 Å in this example), thus eliminating regions of the structures having large conformational differences to compute the superposition parameters. The distances between equivalenced Cá atoms after the minimization procedure are shown in Table 6.

TABLE 6
Distances between equivalent Cá atoms
Molecule 1: HIV-1 PR wt: 1
Molecule 2: HIV-1 PR wt: 2
	Molecule 1	Molecule 2	distance [Å]
	CA PRO 1	CA PRO 1	0.200
	CA GLN 2	CA GLN 2	0.320
	CA ILE 3	CA ILE 3	0.147
	CA THR 4	CA THR 4	0.405
	CA LEU 5	CA LEU 5	0.225
	CA TRP 6	CA TRP 6	0.296
	CA GLN 7	CA GLN 7	0.317
	CA ARG 8	CA ARG 8	0.154
	CA PRO 9	CA PRO 9	0.143
	CA LEU 10	CA LEU 10	0.259
	CA VAL 11	CA VAL 11	0.275
	CA THR 12	CA THR 12	0.307
	CA ILE 13	CA ILE 13	0.207
	CA LYS 14	CA LYS 14	0.273
	CA ILE 15	CA ILE 15	0.434
	CA GLY 16	CA GLY 16	0.469
	CA GLY 17	CA GLY 17	0.414
	CA GLN 18	CA GLN 18	0.319
	CA LEU 19	CA LEU 19	0.161
	CA LYS 20	CA LYS 20	0.155
	CA GLU 21	CA GLU 21	0.196
	CA ALA 22	CA ALA 22	0.338
	CA LEU 23	CA LEU 23	0.246
	CA LEU 24	CA LEU 24	0.292
	CA ASP 25	CA ASP 25	0.142
	CA THR 26	CA THR 26	0.109
	CA GLY 27	CA GLY 27	0.176
	CA ALA 28	CA ALA 28	0.193
	CA ASP 29	CA ASP 29	0.087
	CA ASP 30	CA ASP 30	0.118
	CA THR 31	CA THR 31	0.111
	CA VAL 32	CA VAL 32	0.087
	CA LEU 33	CA LEU 33	0.306
	CA GLU 34	CA GLU 34	0.333
	CA GLU 35	CA GLU 35	0.399
	CA MET 36	CA MET 36	0.296
	CA SER 37	CA SER 37	0.454
	CA LEU 38	CA LEU 38	0.451
	CA PRO 39	CA PRO 39	0.397
	CA GLY 40	CA GLY 40	0.444
	CA ARG 41	CA ARG 41	0.535
	CA TRP 42	CA TRP 42	0.346
	CA LYS 43	CA LYS 43	0.442
	CA PRO 44	CA PRO 44	0.548
	CA LYS 45	CA LYS 45	0.307
	CA MET 46	CA MET 46	0.320
	CA ILE 47	CA ILE 47	0.403
	CA GLY 48	CA GLY 48	0.237
	CA GLY 49	CA GLY 49	0.280
	CA ILE 50	CA ILE 50	0.206
	CA GLY 51	CA GLY 51	0.368
	CA GLY 52	CA GLY 52	0.315
	CA PHE 53	CA PHE 53	0.378
	CA ILE 54	CA ILE 54	0.180
	CA LYS 55	CA LYS 55	0.149
	CA VAL 56	CA VAL 56	0.302
	CA ARG 57	CA ARG 57	0.098
	CA GLN 58	CA GLN 58	0.219
	CA TYR 59	CA TYR 59	0.279
	CA ASP 60	CA ASP 60	0.385
	CA GLN 61	CA GLN 61	0.431
	CA ILE 62	CA ILE 62	0.343
	CA LEU 63	CA LEU 63	0.473
	CA ILE 64	CA ILE 64	0.344
	CA GLU 65	CA GLU 65	0.456
	CA ILE 66	CA ILE 66	0.481
	CA CYS 67	CA CYS 67	0.920
	CA GLY 68	CA CLY 68	0.999
	CA HIS 69	CA HIS 69	0.295
	CA LYS 70	CA LYS 70	0.406
	CA ALA 71	CA ALA 71	0.446
	CA ILE 72	CA ILE 72	0.374
	CA GLY 73	CA GLY 73	0.259
	CA THR 74	CA THR 74	0.276
	CA VAL 75	CA VAL 75	0.165
	CA LEU 76	CA LEU 76	0.220
	CA VAL 77	CA VAL 77	0.202
	CA GLY 78	CA GLY 78	0.231
	CA PRO 79	CA PRO 79	0.131
	CA THR 80	CA THR 80	0.374
	CA PRO 81	CA PRO 81	0.472
	CA VAL 82	CA VAL 82	0.554
	CA ASN 83	CA ASN 83	0.149
	CA ILE 84	CA ILE 84	0.261
	CA ILE 85	CA ILE 85	0.223
	CA GLY 86	CA GLY 86	0.130
	CA ARG 87	CA ARG 87	0.165
	CA ASN 88	CA ASN 88	0.103
	CA LEU 89	CA LEU 89	0.072
	CA LEU 90	CA LEU 90	0.076
	CA THR 91	CA THR 91	0.114
	CA GLN 92	CA GLN 92	0.115
	CA ILE 93	CA ILE 93	0.204
	CA GLY 94	CA GLY 94	0.220
	CA CYS 95	CA CYS 95	0.068
	CA THR 96	CA THR 96	0.185
	CA LEU 97	CA LEU 97	0.095
	CA ASN 98	CA ASN 98	0.311
	CA PHE 99	CA PHE 99	0.216
	CA PRO 101	CA PRO 101	0.455
	CA GLN 102	CA GLN 102	0.121
	CA ILE 103	CA ILE 103	0.120
	CA THR 104	CA THR 104	0.109
	CA LEU 105	CA LEU 105	0.128
	CA TRP 106	CA TRP 106	0.205
	CA GLN 107	CA GLN 107	0.229
	CA ARG 108	CA ARG 108	0.211
	CA PRO 109	CA PRO 109	0.195
	CA LEU 110	CA LEU 110	0.135
	CA VAL 111	CA VAL 111	0.086
	CA THR 112	CA THR 112	0.166
	CA ILE 113	CA ILE 113	0.199
	CA LYS 114	CA LYS 114	0.333
	CA ILE 115	CA ILE 115	0.356
	CA GLY 116	CA GLY 116	0.671
	CA GLY 117	CA GLY 117	0.709
	CA GLN 118	CA GLN 118	0.370
	CA LEU 119	CA LEU 119	0.258
	CA LYS 120	CA LYS 120	0.156
	CA GLU 121	CA GLU 121	0.250
	CA ALA 122	CA ALA 122	0.276
	CA LEU 123	CA LEU 123	0.103
	CA LEU 124	CA LEU 124	0.112
	CA ASP 125	CA ASP 125	0.078
	CA THR 126	CA THR 126	0.057
	CA GLY 127	CA GLY 127	0.121
	CA ALA 128	CA ALA 128	0.098
	CA ASP 129	CA ASP 129	0.190
	CA ASP 130	CA ASP 130	0.302
	CA THR 131	CA THR 131	0.073
	CA VAL 132	CA VAL 132	0.178
	CA LEU 133	CA LEU 133	0.147
	CA GLU 134	CA GLU 134	0.239
	CA GLU 135	CA GLU 135	0.101
	CA MET 136	CA MET 136	0.235
	CA SER 137	CA SER 137	0.391
	CA LEU 138	CA LEU 138	0.364
	CA PRO 139	CA PRO 139	0.532
	CA GLY 140	CA GLY 140	0.213
	CA ARG 141	CA ARG 141	0.448
	CA TRP 142	CA TRP 142	0.133
	CA LYS 143	CA LYS 143	0.195
	CA PRO 144	CA PRO 144	0.082
	CA LYS 145	CA LYS 145	0.359
	CA MET 146	CA MET 146	0.306
	CA ILE 147	CA ILE 147	0.076
	CA GLY 148	CA GLY 148	0.214
	CA GLY 149	CA GLY 149	0.205
	CA ILE 150	CA ILE 150	0.163
	CA GLY 151	CA GLY 151	0.287
	CA GLY 152	CA GLY 152	0.318
	CA PHE 153	CA PHE 153	0.125
	CA ILE 154	CA ILE 154	0.189
	CA LYS 155	CA LYS 155	0.384
	CA VAL 156	CA VAL 156	0.510
	CA ARG 157	CA ARG 157	0.405
	CA GLN 158	CA GLN 158	0.139
	CA TYR 159	CA TYR 159	0.361
	CA ASP 160	CA ASP 160	0.252
	CA GLN 161	CA GLN 161	0.414
	CA ILE 162	CA ILE 162	0.337
	CA LEU 163	CA LEU 163	0.202
	CA ILE 164	CA ILE 164	0.359
	CA GLU 165	CA GLU 165	0.463
	CA ILE 166	CA ILE 166	0.347
	CA CYS 167	CA CYS 167	0.256
	CA GLY 168	CA GLY 168	0.471
	CA HIS 169	CA HIS 169	0.658
	CA LYS 170	CA LYS 170	0.489
	CA ALA 171	CA ALA 171	0.445
	CA ILE 172	CA ILE 172	0.396
	CA GLY 173	CA GLY 173	0.523
	CA THR 174	CA THR 174	0.130
	CA VAL 175	CA VAL 175	0.156
	CA LEU 176	CA LEU 176	0.077
	CA VAL 177	CA VAL 177	0.129
	CA GLY 178	CA GLY 178	0.276
	CA PRO 179	CA PRO 179	0.272
	CA THR 180	CA THR 180	0.580
	CA PRO 181	CA PRO 181	0.436
	CA VAL 182	CA VAL 182	0.328
	CA ASN 183	CA ASN 183	0.180
	CA ILE 184	CA ILE 184	0.151
	CA ILE 185	CA ILE 185	0.104
	CA GLY 186	CA GLY 186	0.059
	CA ARG 187	CA ARG 187	0.058
	CA ASN 188	CA ASN 188	0.183
	CA LEU 189	CA LEU 189	0.164
	CA LEU 190	CA LEU 190	0.051
	CA THR 191	CA THR 191	0.216
	CA GLW 192	CA GLN 192	0.162
	CA ILE 193	CA ILE 193	0.158
	CA GLY 194	CA GLY 194	0.047
	CA CYS 195	CA CYS 195	0.050
	CA THR 196	CA THR 196	0.200
	CA LEU 197	CA LEU 197	0.165
	CA ASN 198	CA ASN 198	0.074

[0123] The distances between the atoms of the inhibitors 1 and 2 to atoms of the protein, that is, hydrogen-bond donors and acceptors, were computed and are shown in Table 7.

TABLE 7
Distances between atoms of inhibitors and atoms of
the proteins
	Wt: 1 complex	Wt: 2 complex
	O2-Wat301	2.92	3.02
	N1-027	3.36	3.58
	O6-N30	3.30	3.50
	06-N29	3.19	3.51
	O7-N29	2.84	—
	O7-OD1 29	3.42	—
	O7-O1	3.31	—
	O3-OD 25 (out) A	2.50	2.80
	O3-OD 25 (in) A	2.65	2.66
	O3-OD 25 (out) B	3.27	3.07
	O3-OD 25 (in) B	2.80	2.68
	O5-Wat301	2.70	2.77
	O8-N 30	3.16	—
	N3-N 30		3.17
	N3-OD2 30		3.15

[0124] Inhibitors 1 (FIG. 1) and 2 (Amprenavir) have similar structural elements, in particular their core, i.e. groups at the P1-P1′60 positions. However, 2 has a THF group while 1 has a bis-THF group at the P2′ position. The P2 groups are identical except for the substitution of an ether oxygen atom in 1 as compared to an amine nitrogen atom at the same position in 2. Table 7 shows that 1 forms more interactions with the atoms of the protein that were previously identified as belonging to the structurally conserved substructure than does compound 2. For example, the O7 oxygen atom in compound 1, that forms an interaction with N29 nitrogen of the protease, has no counterpart in compound 2. Instead, the O6 oxygen atom of 2 forms longer (and presumably weaker) hydrogen bonds with both N30 (3.50 Å) and N29 (3.51 Å). In contrast, the O6 oxygen of compound 1 forms a shorter (and presumably stronger) hydrogen bond with N29 (3.19 Å). Additionally, as can be seen in Table 7, where both compounds 1 and 2 form interactions with atoms in the structurally conserved substructure of HIV protease, the distances between interacting atoms are consistently shorter for compound 1, indicative of presumably stronger binding interactions.

[0125] Examples 1 and 2 were used to identify a three dimensionally-conserved substructure of HIV protease that is involved in the binding of HIV protease inhibitors and, in particular, to identify atoms of these substructural elements that are involved in forming interactions with atoms of HIV protease inhibitors. This substructure is defined by the set of atomic coordinates (in orthogonal coordinates) provided in Table 8 and any equivalent set derived by applying arbitrary rotations and translations to the set of atomic coordinates in Table 8. The values of the coordinates (X,Y,Z) of the atoms defining the substructure are affected by a standard error σ. Therefore (X,Y,Z) values for each atom are those defined in the intervals (X−σ, X+σ) for coordinate X, (Y−σ, Y+σ) for coordinate Y, and (Z−σ, Z+σ) for coordinate Z.

TABLE 8
Three dimensionally-conserved substructure of HIV protease
Atom	X [Å]	Y [Å]	Z [Å]	σ [Å]	Description
Substructure of the protein atoms
Oxygen	−7.9	13.6	27.4	0.5	Oxygen atom of water molecule
					coordinated to main chain amide
					nitrogen atoms of amino acid
					Gly 49 and Gly 149
O27	−13.8	17.7	30.4	0.5	Main Chain carbonyl oxygen
					atom of amino acid Gly 27
N29	−13.4	18.2	34.5	0.5	Main chain amide nitrogen
					atom of amino acid Asp 29
N30	−11.9	18.6	36.7	0.5	Main chain amide nitrogen
					atom of amino acid Asp 30
OD1 25	−11.3	21.2	28.7	0.5	Carboxylate oxygen atom of
					aminoacid Asp 25
OD2 25	−9.4	20.4	29.3	0.5	Carboxylate oxygen atom of
					aminoacid Asp 25
OD1 125	−12.7	20.3	26.4	0.5	Carboxylate oxygen atom of
					aminoacid Asp 125
OD2 125	−12.7	20.3	26.4	0.5	Carboxylate oxygen atom of
					aminoacid Asp 125
N129	−8.9	20.5	20.7	0.5	Main chain amide nitrogen atom
					of amino acid Asp 129
N130	−10.1	19.5	18.6	0.5	Main chain amide nitrogen atom
					of amino acid Asp 130
Substructure of the inhibitor atoms
Hydrogen	−8.8	17.5	25.7	0.5	Interacting with main chain
Bond					carbonyl oxygen atom of amino
donor					acid Gly 27
Atom
Hydrogen	−8.5	15.3	25.1	0.5	Interacting with Oxygen atom of
Bond					water molecule coordinated to
acceptor					main chain amide nitrogen
Atom					atoms of amino acid Gly 49 and
					Gly 149
Hydrogen	−10.4	19.1	27.4	0.5	Interacting with carboxylate
Bond					oxygen atoms of aminoacids
donor-					Asp 25 and Asp 125
acceptor
Atom
Hydrogen	−8.9	14.0	29.8	0.5	Interacting with Oxygen atom of
Bond					water molecule coordinated to
acceptor					main chain amide nitrogen
Atom					atoms of amino acid Gly 49 and
					Gly 149
Hydrogen	−8.6	17.3	20.7	0.5	Main chain amide nitrogen atom
Bond					of amino acid Asp 30
acceptor
Atom
Hydrogen	−6.9	18.7	21.4	0.5	Interacting with main chain
Bond					amide nitrogen atom of amino
acceptor					acid Asp 29
Atom
O8	−10.7	15.8	35.8	0.5	Interacting with main chain
					amide nitrogen atom of amino
					acid Asp 130

EXAMPLE 3

[0126] The following example demonstrates that a protease inhibitor that contains atoms that can make favorable interactions with the atoms of the substructure may exhibit broad spectrum activity.

[0127] Compounds 1 and 3 contain a Bis-THF group at the P2 position that contains two atoms, in particular, hydrogen bond acceptor oxygen atoms, that can form hydrogen bonds with the two hydrogen atoms attached to the backbone amide nitrogen atoms on the protein at residues 29 and 30. Compound 2 is similar to 1 except that 2 contains a THF group at P2 with only a single hydrogen bond acceptor oxygen atom. All three compounds differ in the P2′ substituent. Compounds 1 and 3 both are unaffected by the two active site mutations, V82F and 184V, and Ki values for wild type and mutant enzymes are similar for both compounds. In contrast, compound 2, which contains only a single hydrogen bond acceptor atom in the P2 substitutent, is dramatically affected by the active site mutations, which demonstrate high level resistance to 2.

[0128] The antiviral activity of compounds 1 and 3 against HIV derived from patient isolates that contain multiple mutations are equivalent to their activity against wild type HIV strains. In contrast, compound 2 is much less effective against the same mutant viruses. None of the patients from whom virus was isolated had ever been exposed to any of the compounds tested herein. Nonetheless, compound 2 exhibited cross resistance to these virus strains that is typically seen with all clinically useful HIV protease inhibitors −4 (Saquinavir), 5 (Ritonavir), 6 (Indinavir) and 7 (Nelfinavir). Compounds 2, 4, 5, 6, and 7 have very different chemical structures, but nonetheless behave as a single class with respect to their antiviral behavior against wild type and multidrug resistant HIV strains. All compounds are dramatically less potent against the multidrug resistant strains of HIV.

[0129] In sharp contrast, compounds 1 and 3, which closely resemble each other as well as compound 2, exhibit broad spectrum activity in that they are equally effective against wild type and mutant HIV strains that exhibit high level multidrug resistance towards compounds 2, 4, 5, 6, and 7. The broad spectrum activity of compound 1 was completely unexpected and contrasts with the common and typical loss of antiviral potency experienced with compounds like 2, 4, 5, 6, 7, and indeed most other HIV protease inhibitors represented as similar or different structures that have been reported.

[0130] The development and application of the 3D motif method described above successfully revealed the presence of a unique, three dimensionally-conserved substructure of HIV protease that is useful in the design of broad spectrum inhibitors. Based on this method, compound 3 was predicted, on the basis of comparative molecular modeling using the coordinates of the complexes of compound 1 with wild type and V82F/184V mutant HIV proteases, to be able to make the same key interaction as compound 1 and thereby to exhibit broad spectrum activity. Based on these data, it is feasible to design protease inhibitors that are predicted to have broad spectrum activity, and are predicted to be useful for the treatment of both wild type (first line therapy) and drug resistant (salvage therapy) HIV infections.

EXAMPLE 4

[0131] This example illustrates the method by which experimentally-determined crystal structures of two different target proteins, DHQases, from two different bacterial species can be compared and analyzed for the existence of a three-dimensionally conserved substructure even in the absence of readily discernible or statistically significant sequence similarity. DHQases from different bacterial species typically exhibit less than 30% sequence identity (FIG. 2). A schematic map showing the key interactions of the substrate-based inhibitor, DHQO, with the active site residues for the Type II DHQase from M. tuberculosis is provided in FIG. 3.

[0132] The structures of wild type DHQase from M. tuberculosis and a homologous DHQase from Pseudomonas putidas were determined using conventional x-ray crystallography techniques. The structures were analyzed by means of (a) an overall superposition of the atoms of the protein structures. This analysis requires three dimensional atomic coordinates of the protein structures.

[0133] The superposition of the protein structures was performed in a two step process: 1) the distance between all pairs of corresponding Cá atoms (Cá atom of residue number 1 in one protein to Cá atom of residue number 1 in the second protein; Cá atom of residue number 2 in one protein to Cá atom of residue number 2 in the second protein; and so on) of the polypeptide chains is minimized by means of a least-square algorithm; 2) the superposition is refined by minimizing, in an iterative process, the distances between corresponding Cá atoms that are closer than a given distance (0.4 Å in this example), thus eliminating regions of the structures having large conformational differences to compute the superposition parameters. The distances between equivalenced Cá atoms after the minimization procedure are shown in Table 9.

TABLE 9
Distances between equivalent Cá atoms
Molecule 1: DHQase P. putida wt: qxa
Molecule 2: DHQase M. tuberculosis wt: gt33
	Molecule 1	Molecule 2	distance [Å]
	CA MET 2	CA GLU 2	1.078
	CA ALA 3	CA LEU 3	1.504
	CA THR 4	CA ILE 4	1.800
	CA LEU 5	CA VAL 5	1.283
	CA LEU 6	CA ASN 6	0.911
	CA VAL 7	CA VAL 7	0.715
	CA LEU 8	CA ILE 8	0.298
	CA HIS 9	CA ASN 9	0.211
	CA GLY 10	CA GLY 10	0.591
	CA PRO 11	CA PRO 11	0.599
	CA ASN 12	CA ASN 12	0.487
	CA LEU 13	CA LEU 13	0.428
	CA ASN 14	CA GLY 14	0.229
	CA LEU 15	CA ARG 15	0.685
	CA LEU 16	CA LEU 16	0.541
	CA GLY 17	CA GLY 17	1.693
	CA THR 18	CA ARG 18	2.287
	CA ARG 19	CA ARG 19	2.956
	CA GLN 20	CA GLN 20	3.475
	CA PRO 21	CA PRO 21	3.390
	CA GLY 22	CA ALA 22	4.037
	CA THR 23	CA VAL 23	3.770
	CA TYR 24	CA TYR 24	2.521
	CA GLY 25	CA GLY 25	1.170
	CA SER 26	CA GLY 26	1.642
	CA THR 27	CA THR 27	1.454
	CA THR 28	CA THR 28	1.532
	CA LEU 29	CA HIS 29	1.471
	CA GLY 30	CA ASP 30	1.632
	CA GLN 31	CA GLU 31	1.966
	CA ILE 32	CA LEU 32	1.586
	CA ASN 33	CA VAL 33	1.875
	CA GLN 34	CA ALA 34	2.230
	CA ASP 35	CA LEU 35	2.343
	CA LEU 36	CA ILE 36	1.927
	CA GLU 37	CA GLU 37	2.284
	CA ARG 38	CA ARG 38	2.980
	CA ARG 39	CA GLU 39	2.917
	CA ALA 40	CA ALA 40	2.719
	CA ARG 41	CA ALA 41	3.367
	CA GLU 42	CA GLU 42	3.534
	CA ALA 43	CA LEU 43	3.281
	CA GLY 44	CA GLY 44	3.161
	CA HIS 45	CA LEU 45	2.899
	CA HIS 46	CA LYS 46	1.844
	CA LEU 47	CA ALA 47	1.599
	CA LEU 48	CA VAL 48	1.201
	CA HIS 49	CA VAL 49	2.053
	CA LEU 50	CA ARG 50	1.045
	CA GLN 51	CA GLN 51	0.266
	CA SER 52	CA SER 52	0.300
	CA ASN 53	CA ASP 53	0.282
	CA ALA 54	CA SER 54	0.348
	CA GLU 55	CA GLU 55	0.326
	CA TYR 56	CA ALA 56	0.238
	CA GLU 57	CA GLN 57	0.380
	CA LEU 58	CA LEU 58	0.455
	CA ILE 59	CA LEU 59	0.413
	CA ASP 60	CA ASP 60	0.984
	CA ARG 61	CA TRP 61	1.452
	CA ILE 62	CA ILE 62	1.338
	CA HIS 63	CA HIS 63	1.310
	CA ALA 64	CA GLN 64	2.327
	CA ALA 65	CA ALA 65	2.526
	CA ARG 66	CA ALA 66	3.063
	CA ASP 67	CA ASP 67	3.449
	CA GLU 68	CA
	CA GLY 69	CA ALA 68	2.318
	CA VAL 70	CA ALA 69	1.691
	CA ASP 71	CA GLU 70	0.812
	CA PHE 72	CA PRO 71	0.515
	CA ILE 73	CA VAL 72	0.561
	CA ILE 74	CA ILE 73	0.547
	CA LEU 75	CA LEU 74	0.380
	CA ASN 76	CA ASN 75	0.277
	CA PRO 77	CA ALA 76	0.369
	CA ALA 78	CA GLY 77	0.952
	CA ALA 79	CA GLY 78	0.421
	CA PHE 80	CA LEU 79	0.714
	CA THR 81	CA THR 80	0.575
	CA HIS 82	CA HIS 81	0.142
	CA THR 83	CA THR 82	0.222
	CA SER 84	CA SER 83	0.741
	CA VAL 85	CA VAL 84	0.719
	CA ALA 86	CA ALA 85	0.415
	CA LEU 87	CA LEU 86	0.667
	CA ARG 88	CA ARG 87	0.660
	CA ASP 89	CA ASP 88	0.426
	CA ALA 90	CA ALA 89	0.697
	CA LEU 91	CA CYS 90	1.233
	CA LEU 92	CA ALA 91	1.319
	CA ALA 93	CA GLU 92	2.852
	CA VAL 94	CA LEU 93	4.165
	CA SER 95	CA SER 94	3.605
	CA ILE 96	CA ALA 95	3.840
	CA PRO 97	CA PRO 96	2.414
	CA PHE 98	CA LEU 97	0.314
	CA ILE 99	CA ILE 98	0.251
	CA GLU 100	CA GLU 99	0.095
	CA VAL 101	CA VAL 100	0.131
	CA HIS 102	CA HIS 101	0.318
	CA ILE 103	CA ILE 102	0.117
	CA SER 104	CA SER 103	0.229
	CA ASN 105	CA ASN 104	0.203
	CA VAL 106	CA VAL 105	0.193
	CA HIS 107	CA HIS 106	0.499
	CA LYS 108	CA ALA 107	0.498
	CA ARG 109	CA ARG 108	0.292
	CA GLU 110	CA GLU 109	0.333
	CA PRO 111	CA GLU 110	0.377
	CA PHE 112	CA PHE 111	0.651
	CA ARG 113	CA ARG 112	0.611
	CA ARG 114	CA ARG 113	0.469
	CA HIS 115	CA HIS 114	0.467
	CA SER 116	CA SER 115	0.293
	CA TYR 117	CA TYR 116	0.483
	CA PHE 118	CA LEU 117	0.468
	CA SER 119	CA SER 118	0.367
	CA ASP 120	CA PRO 119	0.676
	CA VAL 121	CA ILE 120	0.445
	CA ALA 122	CA ALA 121	0.334
	CA VAL 123	CA THR 122	0.405
	CA GLY 124	CA GLY 123	0.372
	CA VAL 125	CA VAL 124	0.375
	CA ILE 126	CA ILE 125	0.250
	CA CYS 127	CA VAL 126	0.328
	CA GLY 128	CA GLY 127	0.332
	CA LEU 129	CA LEU 128	0.473
	CA GLY 130	CA GLY 129	0.272
	CA ALA 131	CA ILE 130	0.551
	CA THR 132	CA GLN 131	0.564
	CA GLY 133	CA GLY 132	0.289
	CA TYR 134	CA TYR 133	0.276
	CA ARG 135	CA LEU 134	0.476
	CA LEU 136	CA LEU 135	0.556
	CA ALA 137	CA ALA 136	0.677
	CA LEU 138	CA LEU 137	0.703
	CA GLU 139	CA ARG 138	0.861
	CA SER 140	CA TYR 139	0.876
	CA ALA 141	CA LEU 140	1.330
	CA LEU 142	CA ALA 141	1.529
	CA GLU 143	CA GLU 142	1.492
	CA GLN 144	CA HIS 143	1.738
	CA LEU 145	CA VAL 144	3.487

[0134] Table 9 shows that the two structures are remarkably similar overall despite their low level sequence identity. However, the structures exhibit very large deviations in some regions, and are highly conserved in others. In particular, this analysis reveals that regions of the enzyme are minimally affected by the large number of amino acid sequence substitutions. The regions of the protein structure which are not significantly affected by the amino acid substitutions are defined as structurally conserved regions. In the present example, the substitutions result in localized structural changes in the backbone of DHQase over a wide range, from 0.095-4.165 Å.

[0135] The distances between the strongly interacting atoms of the inhibitor to atoms of the homologous DHQase proteins, that is P. putida wt: qxa and M. tuberculosis wt: gt33 complexes, were computed and they are displayed in Tables 10 and 11, respectively.

TABLE 10
Distances between atoms of the
inhibitor and atoms of the protein
DHQase P. putida wt: gxa
distance [Å]
C6-PRO11 (O)    3.31
C6-ASN12 (CB)   3.10
N7-TYR24 (OH)   2.45
O12-ASN76 (HD2) 2.96
O13-HIS102 (CB) 3.38
O12-SER104 (N)  3.35
C6-ASN12 (CB)   3.10

[0136]

TABLE 11
Distances between atoms of the
inhibitor and atoms of the protein
DHQase M. tuberculosis wt: gt33
distance [Å]
N14-PRO11 (O)     3.35
O15-PRO11 (O)     3.01
O15-LEU 13 (CG)   3.36
O15-ARG 19 (NH1)  3.26
O15-ARG 19 (NH2)  3.32
O7-ASN 75 (OD1)   2.50
O13-ASN 75 (ND2)  2.98
N14-GLY 77 (CA)   3.01
O9-HIS 81 (NE2)   2.82
O7-HIS 101 (ND1)  3.25
O11-ILE 102 (N)   3.33
O13-ILE 102 (N)   2.77
O11-SER 103 (N)   2.96
O11-SER 103 (OG)  2.68
O9-ARG 112 (NH2)  3.09
O10-ARG 112 (NH2) 3.02

[0137] The methods of Examples 1-3 were applied to the DHQase data to identify a three dimensionally-conserved substructure of DHQase that is involved in the binding of DHQase inhibitors, in particular, to identify the relevant target substructure for developing broad spectrum inhibitors. This substructure is defined by the set of atomic coordinates (in orthogonal coordinates) provided in Table 12 and any equivalent set derived by applying arbitrary rotations and translations to the set of atomic coordinates in Table 12. The values of the coordinates (X,Y,Z) of the atoms defining the substructure are affected by a standard error σ. Therefore (X,Y,Z) values for each atom are those defined in the intervals (X−σ, X+σ) for coordinate X, (Y−σ, Y+σ) for coordinate Y, and (Z−σ, Z+σ) for coordinate Z.

TABLE 12
Three dimensionally-conserved substructure of DHQase, M. tuberculosis
Atom	X [Å]	Y [Å]	Z [Å]	σ [Å]	Description
Substructure of the protein atoms
OD1	26.265	68.912	21.219	0.5	Side chain carbonyl
ASN75					oxygen atom of amino
					acid ASN 75
ND2	27.336	66.960	20.951	0.5	Side chain nitrogen
ASN 75					atom of amino acid
					ASN 75
NE2	28.343	76.425	22.111	0.5	Side chain nitrogen
HIS 81					atom of amino acid
					HIS 81
ND1	28.079	70.604	23.662	0.5	Side chain nitrogen
HIS 101					atom of amino acid
					HIS 101
N ILE	31.227	67.167	22.168	0.5	Main chain amide
102					nitrogen atom of
					atom acid ILE 102
N SER	33.754	68.315	21.558	0.5	Main chain amide
103					nitrogen atom of
					amino acid Ser 103
OG SER	33.946	71.059	20.735	0.5	Side chain hydroxyl
103					oxygen atom of amino
					acid SER 103
Substructure of the inhibitor
Hydrogen	29.600	68.554	20.298	0.5	Interacting with main
bond					chain nitrogen atom of
acceptor					ILE 102 and side chain
atom					nitrogen atom of ASN
					75
Hydrogen	28.031	70.739	20.422	0.5	Interacting with side
bond					chain oxygen atom of
donor-					ASN75 and side chain
acceptor					nitrogen atom of
atom					HIS 101
Hydrogen	29.664	74.658	20.493	0.5	Interacting side chain
bond					nitrogen atom of HIS
donor-					81
acceptor
atom
Hydrogen	31.451	69.835	20.531	0.5	Interacting with main
bond					chain nitrogen atom
acceptor					and side chain oxygen
atom					atom of SER 103

Other Embodiments

[0138] All publications and patent applications, and patents mentioned in this specification are herein incorporated by reference.

[0139] While the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications. Therefore, this application is intended to cover any variations, uses, or adaptations of the invention that follow, in general, the principles of the invention, including departures from the present disclosure that come within known or customary practice within the art.

Claims

1. A method for the structure-based design of a drug that can act as an inhibitor of at least two different biological entities, said method comprising the steps of: (a) providing at least one structure of a wild type target protein or an inhibitor-wild type target protein complex; (b) providing at least one structure of a variant target protein or an inhibitor-variant target protein complex; (c) comparing at least one structure from step (a) with at least one structure from step (b) to determine whether there exists a common three-dimensionally conserved substructure comprising the atomic coordinates of the structurally conserved atoms of the inhibitors and structurally conserved atoms of the target proteins; and (d) if a conserved substructure exists, using said atomic coordinates of said conserved substructure to select a compound having atoms matching those of said structurally conserved atoms of the inhibitors, wherein the selection of said compound is performed using computer modeling.

2. A method for the structure-based drug design of a broad spectrum inhibitor, said method comprising the steps of: (a) providing at least one structure of a wild type target protein or an inhibitor-wild type target protein complex; (b) providing at least one structure of a variant target protein or an inhibitor-variant target protein complex; (c) comparing at least one structure from step (a) with at least one structure from step (b) to determine whether there exists a common three-dimensionally conserved substructure comprising the atomic coordinates of the structurally conserved atoms the target proteins or a common three-dimensionally conserved substructure comprising the atomic coordinates of the structurally conserved atoms of the inhibitors and structurally conserved atoms of the target proteins; and (d) if a conserved substructure exists, using said atomic coordinates of said conserved substructure to select a compound having atoms matching those of said structurally conserved atoms of the inhibitors or to design a compound that binds to said target protein, wherein the selection of said compound is performed using computer modeling.

3. The method of claim 1, further comprising the steps of: (e) comparing at least one structure from step (a) with at least one structure from step (b) to determine whether there exists a three-dimensionally non-conserved substructure comprising the atomic coordinates of the structurally non-conserved atoms of the inhibitors and structurally non-conserved atoms of the target proteins; and (f) if a non-conserved substructure exists, using said atomic coordinates of said non-conserved substructure to reject a compound having atoms matching those of said structurally non-conserved atoms of the inhibitors, wherein the rejection of said compound is performed in conjunction with computer modeling.

4. The method of claim 1, wherein at least two structures from step b are used in step c.

5. The method of claim 4, wherein at least four structures from step b are used in step c.

6. The method of claim 4, wherein said target proteins comprise at least two variant forms.

7. The method of claim 6, wherein said target proteins comprise at least four variant forms

8. The method of claim 1, wherein the inhibitors in said inhibitor-wild type target protein complex and said inhibitor-variant target protein complex are the same.

9. The method of claim 1, wherein the inhibitors in said inhibitor-wild type target protein complex and said inhibitor-variant target protein complex are different.

10. The method of claim 1, wherein said inhibitors are competitive inhibitors.

11. The method of claim 1, wherein said inhibitors are noncompetitive inhibitors.

12. The method of claim 1, wherein said inhibitors are reversible inhibitors.

13. The method of claim 1, wherein said inhibitors are irreversible inhibitors.

14. The method of claim 1, wherein said variant target protein is a homologous target protein.

15. The method of claim 1, wherein said variant target protein is a mutant target protein.

16. The method of claim 1, wherein at least one of said structures is a crystal structure.

17. The method of claim 1, wherein at least one of said structures is an nmr structure.

18. The method of claim 1, wherein at least one of said structures is derived using computational methods.

19. The method of claim 1, wherein said target protein is expressed in a microbe and said microbe is selected from the group consisting of viruses, bacteria, protozoa, or fungi.

20. The method of claim 1, wherein said target protein is expressed in a neoplasm.

21. The method of claims 19 or 20, wherein said target protein is selected from the group consisting of an enzyme, a receptor, a structural protein, a component of a macromolecular complex, a component of a metabolic pathway, or an assembly of biological molecules.

22. The method of claim 21, wherein said enzyme is selected from the group consisting of reverse transcriptases, proteases, DNA and RNA polymerases, methylases, oxidases, hydratases, esterases, acyl transferases, helicases, topoisomerases, and kinases.

23. The method of claim 22, wherein said enzyme is HIV protease.

24. The method of claim 23, wherein said inhibitors are selected from the group consisting of indinavir, nelfinavir, ritonavir, saquinavir, amprenavir, lopinavir, and UIC-94003.

25. The method of claim 23, wherein said structurally conserved atoms of the inhibitor and structurally conserved atoms of the protease have the atomic structural coordinates as provided in Table 8.

26. The method of claim 22, wherein said enzyme is 3-dehydroquinate dehydratase.

27. The method of claim 26, wherein said structurally conserved atoms of the 3-dehydroquinate dehydratase have the atomic structural coordinates as provided in Table 12.

28. A compound having a chemical structure selected using the method of claim 19, wherein said compound has broad spectrum activity against wild type and variant microbes.

29. A compound having a chemical structure selected using the method of claim 20, wherein said compound has broad spectrum activity against wild type and variant neoplasms.

30. The compound of claims 28 or 29, wherein said compound has an IC50, variant/IC50, wild type ratio of less than 20.

31. The compound of claim 30, wherein said IC50, variant/IC50, wildtype ratio is less than 6.

32. The compound of claims 28 or 29, wherein said compound has broad spectrum activity against at least 3 mutant biological entities.

33. The compound of claim 28, wherein said compound has broad spectrum activity against at least 3 different organisms expressing homologous target proteins.

34. A pharmaceutical composition comprising a compound of claim 28 and a pharmaceutically acceptable carrier or diluent.

35. A pharmaceutical composition comprising a compound of claim 29 and a pharmaceutically acceptable carrier or diluent.

36. A compound having a chemical structure selected using the method of any one of claims 23-25, wherein said compound has broad spectrum activity against HIV protease.

37. The compound of claim 36, wherein said compound has an IC50, variant/IC50, wild type ratio of less than 10.

38. The compound of claim 37, wherein said IC50, variant/IC50, wild type ratio is less than 6.

39. The compound of claim 36, wherein said compound has broad spectrum activity against at least 3 mutant biological entities.

40. A pharmaceutical composition comprising a compound of claim 36 and a pharmaceutically acceptable carrier or diluent.

41. A compound having a chemical structure selected using the method of claims 26 or 27, wherein said compound has broad spectrum activity against 3-dehydroquinate dehydratase.

42. The compound of claim 41, wherein said compound has an IC50, variant/IC50, wild type ratio of less than 20.

43. The compound of claim 42, wherein said IC50, variant/IC50, wild type ratio is less than 10.

44. The compound of claim 43, wherein said compound has broad spectrum activity against at least 3 mutant biological entities.

45. The compound of claim 41, wherein said compound has broad spectrum activity against at least 3 different organisms expressing homologous target proteins.

46. A pharmaceutical composition comprising a compound of claim 41 and a pharmaceutically acceptable carrier or diluent.

47. A method of treating a microbial infection in a patient, said method comprising the step of administering to said patient a pharmaceutical composition of claim 34 in an amount effective to prevent or treat said infection.

48. A method of treating a neoplasm in a patient in need thereof, said method comprising the step of administering to said patient a pharmaceutical composition of claim 35 in amounts effective to treat said neoplasm.

49. A method of treating an HIV infection in a patient in need thereof, said method comprising the step of administering to said patient a pharmaceutical composition of claim 40 in amounts effective to treat said infection.

50. A method of treating a bacterial infection in a patient in need thereof, said method comprising the step of administering to said patient a pharmaceutical composition of claim 46 in amounts effective to treat said infection.

51. The method of claim 50, wherein said bacterial infection is caused by a bacterium selected from the group consisting of C jejuni, V. cholerae, Y pestis, B. anthracis, P. putidas, and M. tuberculosis.